Recent biological studies have shown that a variety of peptides, proteins and phosphoproteins from very diverse tissues, glandular secretions and physiological fluids can bind calcium and inhibit calcium phosphate precipitation. Presently, it is not clear that these are related events. We have shown that human saliva exists as a supersaturated but stable solution of calcium phosphate through the presence of two types of phosphoproteins. The complete structure of one of these (statherin) and the NH2-terminal sequence of the other type of phosphoprotein, a group of four related anionic proline-rich proteins, PRP-I, II, III, and IV provide the basis for understanding the mechanism of protein-solute interaction and calcium binding and represent the first step in the localization of the active site of these inhibitors. The first objective of the proposed project will be the complete sequence determination of the human salivary anionic proline-rich proteins PRP-I, II, III, and IV and comparative sequence analyses on the protein inhibitors in saliva of other animals and other mammalian tissues to determine similar structural features in these molecules. The second objective of the proposed project will focus on the elucidation of the calcium binding and precipitation-inhibition site in the human salivary proteins. This will be attempted by cleavage of the native molecule, purification of peptide fragments and sequence determination of these regions. Localization of the site will be followed by synthesis of active peptide segments and peptide analogues of statherin and the anionic proline-rich proteins. A more long term objective of the proposed research project will include conformational analyses on the human salivary protein inhibitors, synthetic peptides of the inhibitory and/or calcium binding regions and their peptide analogues using fluorescence spectroscopy and multinuclear resonance spectroscopy. These latter studies should provide an accurate understanding of the mechanism of protein-calcium binding.